Reverse transcriptase fidelity and HIV-1 variation.
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Anti-HIV Agents↗
Biomedical subjects
J Balzarini
Publications and source records attributed to J Balzarini.
Novel carbocyclic nucleosides containing a cyclopentyl ring. Adenosine and uridine analogues.
cis-3-Aminomethylcyclopentylmethanol (4) was used as a precursor in the synthesis of carbocyclic nucleosides containing adenine, hypoxanthine, 8-azahypoxanthine, uracil, and 5-iodouracil bases. None of these compounds had appreciable activity against eighteen viruses in the concentration ranges tested. Two of them showed a weak cytostatic activity against three tumor lines.
Adenosine↗
Inhibition of replication of HIV in primary monocyte/macrophages by different antiviral drugs and comparative efficacy in lymphocytes.
Several anti-HIV drugs acting on different steps of virus replication were tested in our experimental model of primary monocyte/macrophages; the results were compared with the activity found in lymphocytes. Nucleoside analogues (AZT, ddI, ddC, d4T, PMEA, 3TC etc.) show greater activity in macrophages (M/M) than in lymphocytes. In particular, the EC50 of AZT, ddC, and ddI in M/M is 2- to 100-fold lower than that found in lymphocytes. This greater efficacy of nucleoside analogues in M/M depends on the enhancement of their chain-terminating activity by the low levels of endogenous deoxynucleoside-triphosphates (dNTP) usually found in resting cells such as M/M. Non-nucleoside reverse transcriptase inhibitors (NNRTI) do not act as chain terminators (thus their antiviral effect is not related to the intracellular concentrations of dNTP); as a consequence the activity of TSAO, HEPT, TIBO, and other NNRTI tested in M/M is similar to that found in lymphocytes. Regarding inhibitors of binding and fusion of HIV, we found that their anti-HIV activity is markedly decreased (or even nullified) when M/M are treated with cytokine activators of M/M function and enhancers of HIV replication. More relevant from a clinical standpoint, protease inhibitors are able to inhibit HIV replication in chronically infected macrophages (i.e., cells carrying the proviral genome already integrated in the host genome). All other inhibitors of late stage of virus life cycle tested (antisense-rev, anti-tat, interferon-alpha and -gamma, phosphorothioate analogues, GLQ-223, etc.) were totally inactive in chronically infected macrophages. The different effects of various classes of HIV inhibitors in lymphocytes and macrophages suggests that AIDS therapy should consider all aspects of the pathogenesis of HIV infection and must be restricted to drugs, or combinations of drugs, active against both lymphocytes and M/M in all body compartments where the virus hides and replicates.
Anti-HIV Agents↗
Red blood cells mediated delivery of 9-(2-phosphonylmethoxyethyl)adenine to primary macrophages: efficiency metabolism and activity against human immunodeficiency virus or herpes simplex virus.
Red blood cells (RBC) may act as selective carriers of drugs to macrophages, an important reservoir of viruses such as human immunodeficiency virus (HIV) and herpes simplex virus type 1 (HSV-1). We therefore assessed the incorporation of 9-(2-phosphonylmethoxyethyl)adenine (PMEA), a potent inhibitor of HIV and HSV-1) into RBC, its delivery to macrophages and its activity against HIV or HSV-1. Loading of PMEA in artificially aged opsonized RBC affords significant levels of intracellular PMEA. RBC metabolize PMEA to its active congener PMEA-diphosphate, although with low efficiency. Exposure of macrophages to RBC-encapsulated PMEA inhibits the replication of both HIV and HSV-1 (about 90% inhibition at the highest RBC:macrophages ratios) even if RBC were removed before virus challenge. By contrast, the antiviral activity of free PMEA removed before virus challenge was irrelevant at concentrations up to 150-fold higher than the 50% effective concentration (EC50). Finally, the antiviral effect of RBC-encapsulated PMEA correlates with PMEA levels in macrophages about 500-fold higher than those achieved by free PMEA (at concentrations 10-fold higher than the EC50). The efficacy of RBC-mediated delivery to macrophages of PMEA (and perhaps of compounds with shorter intracellular half-lives) warrants further studies in infectious diseases involving phagocytizing cells as main targets of the pathogen.
Adenine↗
Phosphoramidate derivatives of d4T as inhibitors of HIV: the effect of amino acid variation.
Phosphoramidate derivatives of the nucleoside analogue, 2',3'-dideoxy-2',3'-didehydro thymidine (d4T) have been prepared as potential membrane-soluble pro-drugs of the big-active free phosphate forms. In particular phenyl phosphates, linked via nitrogen to methyl-esterified amino acids, were studied. All compounds were fully characterised by a range of methods (high-field multinuclear NMR, mass spectrometry and high performance liquid chromatography (HPLC)) and were subjected to in vitro evaluation of their anti-HIV efficacy. The nature of the amino acid appeared to be extremely important for the eventual antiviral action. Of the amino acids studied, L-alanine was the most efficacious, whilst L-proline and glycine were particularly poor. However, an unnatural amino acid moiety, dimethylglycine, could substitute for alanine with little or no loss of activity.
Amides↗
Anti-HIV activity of N-1-adamantyl-4-aminophthalimide.
The discovery of new leads acting via novel modes of action in the treatment of the human immunodeficiency virus (HIV), the causative agent of AIDS, remains a challenge. Along this line we synthesized and evaluated a series of N-substituted 4-aminophthalimides which were designed according to the models of thalidomide, phenytoin (PHT) and ameltolide. From a series of 24 compounds only N-1-adamantyl-4-aminophthalimide was endowed with anti-HIV-1 and -HIV-2 activity in CEM cell cultures.
Adamantane↗
Antiviral and tumor cell antiproliferative SAR studies on tetracyclic eudistomins. II.
In a search for the minimum pharmacophore of the naturally occurring tetracyclic eudistomins, five structural analogues (4-8) were evaluated for their in vitro antiviral and tumor cell antiproliferative activities. For the synthesis of these derivatives both intra- and intermolecular Pictet-Spengler reactions have been used. Opening of the beta-carboline annulated 7-membered D-ring in 6 and 7 resulted in a complete loss of activity. On the other hand, replacement of either the oxygen atom or the sulfur atom in the 7-membered ring by a methylene group in 5 and 8, respectively, is allowed. These results combined with previous SAR data underline the crucial importance of the D-ring in eudistomins as a scaffold for the correct positioning of both basic nitrogen atoms. Also bioisosteric replacement of the bicyclic indole system with a dimethoxyphenyl group, to give the isoquinoline skeleton, is allowed. The tricyclic isoquinoline derivative 4 is, so far, the most promising antiviral analogue; it combines a high potency (MIC at 100 ng/ mL (340 nM)) with high MCC/MIC ratios (ranging from 1000 to 5000 against HSV-1, HSV-2, vaccinia virus, and vesicular stomatitis virus.
Animals↗
Palladium-catalyzed synthesis of [E]-6-(2-acylvinyl)uracils and [E]-6-(2-acylvinyl)-1-[(2-hydroxyethoxy)methyl]uracils--their antiviral and cytotoxic activities.
[E]-6-(2-Acylvinyl)uracils and their corresponding 1-(2-hydroxyethoxy)methyl derivatives were synthesized through palladium-catalyzed reactions which involved an interesting rearrangement. Some of the acylvinyl uracils (3, 4, and 5) and the acyclonucleosides (8 and 10) showed pronounced activity against human T-lymphocyte Molt 4/C8 and CEM cells. However, they were less toxic to murine L1210 and FM3A cells. The compounds did not have any marked antiviral activity.
Animals↗
Varicella-zoster virus thymidine kinase gene and antiherpetic pyrimidine nucleoside analogues in a combined gene/chemotherapy treatment for cancer.
Ten pyrimidine nucleoside analogues, including (B)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and closely related analogues, were evaluated for their cytostatic activity against human osteosarcoma cells transfected with the varicella-zoster virus (VZV) thymidine kinase (tk) (ATP:thymidine 5' phosphotransferase, EC 2.7.2.21) gene. (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil (BVaraU), (E)-5-(2-iodovinyl)-2'-deoxy-2'-fluoro-1-beta-D-arabinofuranosyluracil (IVFAU) and (E)-5-(2-bromovinyl)-2'-deoxy-4'-thiouridine (S-BVDU) were among the most potent inhibitors of VZVtk gene-transfected cell proliferation. They displayed an inhibitory activity at drug concentrations that were up to four orders of magnitude lower than those required to inhibit the corresponding nontransfected tumor cells. Inhibition of cellular DNA polymerase and/or incorporation of the drugs into cellular DNA may be a likely target for the cytostatic activity of the BVDU derivatives against the VZVtk gene-transfected tumor cells. These compounds were approximately 40- to 80-fold more potent cytostatic agents in VZVtk gene-transfected cells than the anti-VZV compound 6-methoxy-9-beta-D-arabinofuranosylpurine (araM), and at least five- to 50-fold more cytostatic than ganciclovir in HSV-1tk gene-transfected murine mammary carcinoma FM3A cells. In addition, the intrinsic resistance of BVaraU, IVFAU and S-BVDU to glycosidic bond cleavage by mammalian dThd phosphorylases makes them promising candidate compounds for the treatment of VZVtk gene-transfected tumors in vivo.
Antiviral Agents↗
Zidovudine-resistant human immunodeficiency virus type 1 strains subcultured in the presence of both lamivudine and quinoxaline HBY 097 retain marked sensitivity to HBY 097 but not to lamivudine.
Replication of zidovudine-resistant human immunodeficiency virus type 1 (HIV-1) strains (containing the 41 Met-->Leu and 215 Thr-->Tyr mutations in reverse transcriptase [RT]) was inhibited to a significantly greater extent by the combination of lamivudine and quinoxaline HBY 097 than by either drug alone or even fully suppressed by concomitant HBY 097 and lamivudine administration at relatively low concentrations. The virus recovered after exposure to the drug combinations individually had acquired the 103 Lys-->Arg, 138 Glu-->Lys, 184 Met-->Ile, and 189 Val-->Ile mutations in the genetic zidovudine-resistance background of zidovudine-resistant HIV-1. These mutants retained marked sensitivity to HBY 097. The genotypic zidovudine-resistance mutations were maintained in the mutant virus RT genomes, and the viruses also remained phenotypically resistant to zidovudine. Given the exquisite potency of the combination of lamivudine and HBY 097 in suppressing viral replication, this combination should be further pursued in clinical trials examining treatment of HIV-1-infected persons.
Anti-HIV Agents↗
SRR-SB3, a disulfide-containing macrolide that inhibits a late stage of the replicative cycle of human immunodeficiency virus.
From a series of macrocyclic diamides possessing the disulfide linkage, only SRR-SB3, a compound that complexes with zinc, was found to inhibit human immunodeficiency virus type 1 (HIV-1; strain IIIB) replication at a concentration of 1.8 to 6.5 micrograms/ml in MT-4, CEM, and peripheral blood mononuclear cells. SRR-SB3 was toxic to MT-4 cells at a concentration of 15.9 micrograms/ml, resulting in a selectivity index of 9 in these cells. This macrolide was also effective against various other HIV-1 strains, including clinical isolates and HIV-1 strains resistant to protease inhibitors and nucleoside and nonnucleoside reverse transcriptase inhibitors. It was also active against various HIV-2 strains, simian immunodeficiency virus (strain MAC251), and Moloney murine sarcoma virus, but not against viruses other than retroviruses. In addition, the compound was found to inhibit chronic HIV-1 infections in vitro. The compound in combination with other antiviral agents, such as zidovudine, zalcitabine, and stavudine, showed an effect that was between additive and synergistic. Time-of-addition experiments indicated that SRR-SB3 acts at a late stage of the HIV-1 replicative cycle.
Animals↗
Antiretroviral activities of acyclic nucleoside phosphonates [9-(2-phosphonylmethoxyethyl)adenine, 9-(2-phosphonylmethoxyethyl)guanine, (R)-9-(2-phosphonylmethoxypropyl)adenine, and MDL 74,968] in cell cultures and murine sarcoma virus-infected newborn NMRI mice.
From a side-by-side comparative study, the acyclic nucleoside phosphonates (R)-9-(2-phosphonylmethoxypropyl)adenine [(R)-PMPA] and 9-(2-methylidene-3-phosphonomethoxypropyl)guanine (MDL 74,968) proved more selective in their inhibitory effect on human immunodeficiency virus types 1 and 2, feline immunodeficiency virus, and Moloney murine sarcoma virus (MSV) in cell cultures than the 9-(2-phosphonylmethoxyethyl) derivatives of adenine (PMEA) and guanine (PMEG). In particular, PMEG proved quite toxic. PMEA, (R)-PMPA, and MDL 74,968 afforded a marked delay in MSV-induced tumor initiation in MSV-infected newborn NMRI mice and substantially delayed associated animal death at doses as low as 4 to 10 mg/kg of body weight. Treatment of the NMRI mice with PMEA, (R)-PMPA, and MDL 74,968 at 25 or 50 mg/kg resulted in a high percentage of long-term survivors.
Adenine↗
Characteristics of the Pro225His mutation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase that appears under selective pressure of dose-escalating quinoxaline treatment of HIV-1.
Treatment of human immunodeficiency virus type 1 (HIV-1)-infected CEM cell cultures with escalating concentrations of the quinoxaline S-2720 resulted in an ordered appearance of single and multiple mutant virus strains that gradually became resistant to the quinoxaline and other nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs). A novel mutation, Pro225His, consistently appeared in a Val106Ala RT-mutated genetic background. The contribution of this mutation to the resistance of the mutant HIV-1 RT to NNRTIs was additive to the resistance caused by the Val106Ala mutation. Interestingly, site-directed mutagenesis studies revealed that the Pro225His-mutated RT had acquired markedly greater sensitivity to bis(heteroaryl)piperazine (BHAP U-90152) (delavirdine) but not to any of the other NNRTIs. The kinetics of inhibition of the Pro225His mutant RT by the NNRTIs (including BHAP U-90152) was not substantially different from that observed for the wild-type RT. The hypersensitivity of the mutant enzyme and virus to BHAP U-90152 could be rationally explained by the molecular-structural determinants of the RT-BHAP complex, which has recently been resolved by X-ray crystallography.
Amino Acid Substitution↗
New neplanocin analogues. VIII. Synthesis and biological activity of 6'-C-ethyl, -ethenyl, and -ethynyl derivatives of neplanocin A.
This report describes the synthesis and antiviral effects of (6'R)-6'-C-ethynyl, -ethenyl, and -ethyl derivatives of neplanocin A (7a, 8a, and 9a, respectively) and the corresponding 6'S-diastereomers (7b, 8b, and 9b, respectively), as examples of 6'-C-substituted analogues of neplanocin A. Grignard reaction of the 6'-formyl derivative 4, which was readily prepared from neplanocin A, with ethynylmagnesium bromide gave a diastereomeric mixture of the corresponding 1,2-addition products 5a and 5b. After removal of the protecting groups, (6'R)- and (6'S)-6'-C-ethynylneplanocin A's (7a, 7b) were separated. The corresponding ethenyl derivatives 8a and 8b and ethyl derivatives 9a and 9b were prepared by catalytic hydrogenation of 7a and 7b, respectively. As compared to neplanocin A, the new neplanocin A derivatives were much weaker inhibitors of S-adenosyl-L-homocysteine hydrolase, the R-diastereomers being more inhibitory than the S-diastereomers. The decreasing order of activity was 7a > 8a > 7b > 9a > 8b > 9b. The cytotoxicity (for CEM cells) followed exactly the same order. Of these compounds, (6'R)-6'-C-ethynylneplanocin A (7a, RENPA) showed an antiviral activity spectrum that was comparable to, and an antiviral specificity that was higher than, that of neplanocin A. RENPA was particularly active against those viruses (i.e. vaccinia virus, vesicular stomatitis virus) that are known to be highly sensitive to AdoHcy hydrolase inhibitors.
Adenosine↗
Synthesis and cytotoxic evaluation of some carbohydrazones and thiocarbohydrazones of various unsaturated ketones and related Mannich bases.
A number of 1-aryl-1-penten-3-ones 1 and related Mannich bases 2 were synthesized and converted either directly or indirectly to the corresponding biscarbohydrazones 3 and 4. Reaction of representative unsaturated ketones with thiocarbohydrazide led to the formation of only the monothiocarbohydrazones 5. The cytotoxicity of these compounds against murine P388 and L1210 cells, human T-lymphocytes and human tumour cell lines was undertaken in order to ascertain their bioactivity and review these data in the light of the theory of sequently cytotoxicity whereby the carbohydrazones would be predicted to be more than twice as cytotoxic as the ketones and Mannich bases from which they were derived. In general, conversion of 1 into 3 was accompanied by a lowering of cytotoxicity. On the other hand, in general the compounds in series 4 were significantly more potent cytotoxic agents than the analogues 2; in fact, some compounds displayed activity comparable with or exceeding that of melphalan. Thus the biscarbohydrazones 4 represent a novel group of cytotoxic agents serving as lead molecules for subsequent development.
Animals↗
[Comparative investigation of the efficacy of two nucleocapsid analogs in FIV infected cats].
Antiviral activity of the acyclic nucleoside phosphonates 9-(2-phosphonylmethoxyethyl)adenine (PMEA) and 9-(3-fluoro-2-phosphonylmethoxypropyl)adenine (FPMPA) against feline immunodeficiency virus (FIV) was investigated in field cats. The study was designed as a placebo-controlled double-blind study enrolling 27 FIV infected cats. Nine cats received PMEA at a dosage of 10 mg/kg body weight, nine cats received FPMPA at a dosage of 25 mg/kg body weight. A variety of parameters were established to evaluate the therapeutic efficacy of the compounds. The improvement was monitored by different aspects: clinical status, index of Karnofsky modified for the cat, laboratory parameters, immunological parameters, surrogate markers, and a virological parameter. Concerning the clinical and immunological parameters cats of both treatment groups disclosed a relevant improvement. Efficacy of antiviral treatment was a little bit higher in the cats treated with PMEA than in the animals injected with FPMPA. However, side effects were also more prominent in the PMEA treated group.
Adenine↗
Concomitant combination therapy for HIV infection preferable over sequential therapy with 3TC and non-nucleoside reverse transcriptase inhibitors.
Exposure to 3TC of HIV-1 mutant strains containing non-nucleoside reverse transcriptase inhibitor (NNRTI)-specific mutations in their reverse transcriptase (RT) easily selected for double-mutant viruses that had acquired the characteristic 184-Ile mutation in their RT in addition to the NNRTI-specific mutations. Conversely, exposure of 3TC-resistant 184-Val mutant HIV-1 strains to nine different NNRTIs resulted in the rapid emergence of NNRTI-resistant virus strains at a time that was not more delayed than when wild-type HIV-1(IIIB) was exposed to the same compounds. The RTs of these resistant virus strains had acquired the NNRTI-characteristic mutations in addition to the preexisting 184-Val mutation. Surprisingly, when the 184-Ile mutant HIV-1 was exposed to a variety of NNRTIs, the 188-His mutation invariably occurred concomitantly with the 184-Ile mutation in the HIV-1 RT. Breakthrough of this double-mutant virus was markedly accelerated as compared with the mutant virus selected from the wild-type or 184-Val mutant HIV-1 strain. The double (184-Ile + 188-His) mutant virus showed a much more profound resistance profile against the NNRTIs than the 188-His HIV-1 mutant. In contrast with the sequential chemotherapy, concomitant combination treatment of HIV-1-infected cells with 3TC and a variety of NNRTIs resulted in a dramatic delay of virus breakthrough and resistance development.
Base Sequence↗
Acyclic nucleotide analogs derived from 8-azapurines: synthesis and antiviral activity.
Reaction of phosphoroorganic synthons with 8-azaadenine, 8-aza-2, 6-diaminopurine, and 8-azaguanine using cesium carbonate yielded regioisomeric 8-azapurine N7-, N8-, and N9-(2-(phosphonomethoxy)alkyl) derivatives. This reaction followed by deprotection afforded isomeric 2-(phosphonomethoxy)ethyl (PME), (S)-(3-hydroxy-2-(phosphonomethoxy)propyl) [(S)-HPMP], (S)-(3-flouro-2-(phosphonomethoxy)propyl) [(S)-FPMP], (S)-(2-(phosphonomethoxy)propyl) [(S)-PMP], and (R)-(2-(phosphonomethoxy)propyl) [(R)-PMP] derivatives. 13C NMR spectra were used for structural assignment of the regioisomers. None of the 8-isomers exhibited any antiviral activity against herpesviruses, Moloney murine sarcoma virus (MSV), and/or HIV. 9-(S)-HPMP-8-azaadenine (23) and PME-8-azaguanine (65) were active against HSV-1, HSV-2, and CMV at 0.2-7 micrograms/mL, VZV at 0.04-0.4 microgram/mL, and MSV (at 0.3-0.6 microgram/mL). PME-8-azaguanine (65) and (R)-PMP-8-azaguanine (71a) protected MT-4 and CEM cells against HIV-1- and HIV-2-induced cytopathicity at a concentration of approximately 2 micrograms/mL.
3T3 Cells↗